The goal of studies in Project II is to define the safety (in vitro cytotoxicity, in vivo toxicity, and immunogenicity) and mechanisms of action that underlie the anti-HIV-1 activities of compounds collectively classified as polybiguanides (PBGs). The hypotheses that form the basis of Project II are that (i) PEHMB-based compounds (or combinations containing PEHMB) can be designed, synthesized, and formulated with little or no toxicity or immunogenicity, and (ii) the mechanism of action of PEHMB against HIV-1 infection involves direct, specific interactions with HIV-1 co-receptors CXCR4 and CCR5. Our studies have identified polyethylene hexamethylene biguanide (PEHMB) as a prototypical PBG with low cytotoxicity, substantial anti-HIV-1 activity, and an in vitro therapeutic index in excess of 1400. The PEHMB molecule will be used as a candidate microbicide as well as the cornerstone for logical, computer-aided design and synthesis strategies directed toward optimizing the efficacy of PEHMB derivatives. Our efforts, which will culminate in demonstrations of in vivo efficacy against HIV-1 in animal models of HIV-1 infection (Project III), will serve as a foundation for clinical trials of highly efficacious anti-HIV-1 PBG-based compounds as safe and effective topical vaginal microbicides. The specific aims of Project II are to (i) assess the effect of PEHMB and derivative compounds, combinations, and formulations (identified in Projects I and III) on the in vitro viability of cells of epithelial and immune origin found in the human female reproductive tract, and on the viability and integrity of vaginal and cervical epithelial tissues using mouse and rabbit models of in vivo toxicity; (ii) demonstrate the impact of PEHMB and select derivative compounds on gene expression patterns and cytokine/chemokine signaling in single cell populations and on immune cell recruitment and inflammatory processes within vaginal and cervical epithelial tissues in the mouse model of in viv0 toxicity (as assessed by microarray analyses and laser capture microdissection); and (iii) determine the effects of interactions between PEHMB and HIV-1 co-receptors CXCR4 and CCR5 on HIV-1 binding and entry events that precede HIV-1 infection and on co-receptor functions. This project is an integral part of highly collaborative studies focused on the development of safe and effective PBG-based microbicides with activity against HIV-I. These studies will serve to significantly advance our development of an affordable topical PBG-based microbicide used for the global reduction or elimination of HIV-1 sexual transmission.